This invention relates to buffering systems for controlling the pH of aqueous media and, more particularly, to pH buffering systems useful in isoelectric focusing and in other electrophoretic processes.
Effectively controlling the pH of aqueous media with suitable biologically acceptable buffers is essential for much of biochemistry and microbiology, as well as other life sciences. It is essential for such biological techniques as, for example, protein extraction, preservation and purification, chromatography and HPLC, enzyme assays and reactions, tissue culture, organ preservation and perfusion, and electrophoresis. Electrophoretic processes present special buffering problems, since the buffering system must be functionally stable under the influence of an electric field. This is particularly so in the case of isoelectric focusing ("IEF"), an electrophoretic technique requiring functionally stable pH gradients.
IEF is a high resolution method useful for both analytical and preparative separation and purification of various biological materials, mainly proteins and peptides, but occasionally also nucleic acids, viruses, and even some living cells or cell organelles. The principle of IEF is based on the fact that certain biomaterials are amphoteric in nature, i.e., are positively charged in acidic media and negatively charged in basic media. At a particular pH value, known as the isoelectric point, they undergo a reversal of net charge polarity passing through a point of zero net charge. The isoelectric point of any given amphoteric substance is a characteristic of its chemical composition. When a mixture of such substances is exposed to an applied external d.c. electric field within a pH gradient, each component of the mixture will migrate electrophoretically until it reaches the pH corresponding to its isoelectric point, where it will become virtually immobilized due to having acquired zero net charge. The end result is a steady state in which all components are focused into narrow pH zones corresponding to their respective isoelectric points.
The effectiveness of IEF as a high resolution separation and purification technique depends heavily on the use of buffering systems which are capable of forming pH gradients exhibiting functional stability under electrophoretic conditions. Two distinct approaches have been taken in the prior art in the development of suitable buffering systems for forming functionally-stable pH gradients. These two approaches differ in the manner in which the pH gradient is generated. In one approach, known as "natural" pH gradients, the pH gradient is generated within the focusing system by the electric field itself. In the other approach, known as "precast" pH gradients, the pH gradient is preformed or precast in the system and not generated by the electric current.
The formation of natural pH gradients relies on buffering systems composed of ampholytes having sharply defined isoelectric points. The theory of this approach is that in an electric field, each ampholyte will migrate electrophoretically towards its isoelectric point, modifying at the same time the local pH. Finally, a steady state will be achieved where all ampholytes have migrated to their isoelectric points, thereby establishing a pH gradient. An ampholyte's ability to focus sharply to its isoelectric point and to effectively control the pH is related to its pK spread, i.e., the spread between its acidic function dissociation constant pK.sub.1 and its basic function dissociation constant pK.sub.2 which are proximal to its isoelectric point pI, the pI being the algebraic midpoint of the spread.
Svensson (Acta Chem. Scand. Vol. 15, pp. 325-341, 1961) was the first to propose this method of gradient formation, but concluded that only ampholytes with a pK spread of less than 3-4 pH units are "good" for this purpose. Unfortunately, most simple ampholytes, such as amino acids and their derivatives, have larger pK spreads and are unusable for formation of natural pH gradients, having neither sufficient buffering power nor electrical conductivity when focused. Consequently, ampholytes having a pK spread of greater than 4 pH units have not previously been found suitable for use in pH gradient formation, thereby eliminating most of the simple ampholytes of known chemical composition. This includes also the so-called Good's buffers (described more fully in Good et al., Biochemistry Vol. 5, p.467, 1966 and Ferguson and Good, Anal. Biochem. Vol. 104, p. 300, 1980) specifically designed for biologically acceptable buffering. Subsequent literature on IEF is too extensive to be cited, but a recent and thorough review of the main electrophoretic methods is to be found in the monograph "The Dynamics of Electrophoresis" by Mosher, Thormann and Saville (VCH Publishers, 1992).
To overcome this obstacle, suitable synthetic buffering mixtures known as carrier ampholytes were formulated as random polymerization products comprising a large number of synthetic polyelectrolytes with a broad range of isoelectric points. Commercially available carrier ampholytes include, for example, those sold under the tradenames "Ampholine", "Pharmalyte", "Servalyte" and "Biolyte". These carrier ampholytes serve well for analytical IEF, since they create stable broad or narrow range pH gradients under electrophoretic conditions, particularly when used in conjunction with stabilizing hydrophilic gel media, such as polyacrylamide or agarose. They are less well suited for preparative IEF, since by the very nature of their synthesis they are chemically ill-defined and thus contaminate the final product. This contamination problem is even further complicated when a gel medium is used for stabilization of the pH gradient. As a result, there have been numerous attempts to formulate natural pH gradient-forming buffer mixtures of known structure. These have included mixtures of ten buffering components, as described by Chrambach et al, U.S. Pat. No. 4,139,440, issued Feb. 13, 1979; and mixtures of at least four buffering components as described by Hearn et al, U.S. Pat. No. 4,279,724, issued Jul. 21, 1981. However, none of these buffer mixtures has proven to be as effective as the carrier ampholytes for forming natural pH gradients over a wide range of pH values.
The second approach to focusing relies on precast pH gradients having the requisite functional stability. This approach has heretofore been limited almost exclusively to employing precast pH gradients wherein the buffering components are immobilized by copolymerization within polyacrylamide gel media or polyacrylamide membranes. Specially formulated copolymerizable buffering agents are commercially available under the tradename "Immobiline", and have been used in the gel-immobilized form primarily for analytical IEF, as well as in the membrane-immobilized form better suited for preparative IEF (cf., for example, Faupel & Righetti, U.S. Pat. No. 4,971,670, issued Nov. 20, 1990.
Thus, until now, the only known effective technique for obtaining gel-free pH gradients useful over a wide range of pH values, has been through natural pH gradient formation, which limits buffering component selection to only those ampholytes which focus sharply to isoelectric points coinciding with the desired buffering pH value. Excluded from this limited selection are most of the simple ampholytes of known chemical composition, which have generally been regarded as unsuitable for use in pH gradient formation.
A number of two-component buffering systems, both real and hypothetical, employing simple ampholytes, weak acids and weak bases, have been proposed for use in pH gradient formation in a series of papers co-authored by the present inventor. These papers include Palusinski et al, Biophysical Chemistry, vol.13, pp. 193-202, (1981); Bier et al, Journal of Chromatography, vol. 211, pp. 313-335 (1981); Bier et al, Science, vol. 219, pp. 1281-1287 (1983); Bier et al, in: Hirai (Ed.) Electrophoresis '83, de Gruyter, Berlin, pp. 99-107 (1984); Mosher et al, Electrophoresis, vol. 6, pp. 545-551 (1985); and Bier et al, Journal of Chromatography, vol. 604, pp. 73-83 (1992). The buffering systems disclosed in these papers have had only very limited utility, either in purely theoretical applications or in providing buffering in a limited pH range at or near neutrality. Furthermore, these papers fail to establish any criteria for the rational selection of buffering components to provide buffer pairs having practical utility over a wide range of pH values. In fact, out of all the various buffer pairs disclosed in these six papers, only two would actually meet the selection criteria of the present invention. One of these is a hypothetical buffer pair of a first ampholyte whose pK.sub.2 is 8.0 and a second ampholyte whose pK.sub.1 is 5.0, disclosed in the 1981 Palusinski et al paper (Table 2, System C). The other is the buffer pair of alpha-Asp-His/isoglutamine, disclosed in the 1981 Bier et al paper as the fourteenth of twenty-eight entries in Table II. Moreover, from a reading of these papers, one would not expect either of these two buffer pairs to have any particular utility other than in theoretical studies. In fact, both of these buffer pairs are indicated in the papers as being less desirable than other listed buffer pairs for the purposes described. Thus, the listing of these two buffer pairs would not in any way be suggestive of the selection criteria utilized in carrying out the present invention.